Natural killer cells express cell surface receptors encoded by genes of the Ly49 family, specific for class I molecules, engagement of which down- regulates NK lysis of target cells. These receptors apparently protect normal cells from lysis but allow the lysis of cells that repress expression of one or more class I molecules. The receptors discriminate, to some extent, the products of different class I alleles and loci. Different receptor family members are expressed by distinct but overlapping NK cell populations. Hence there is a repertoire of NK specificities for class I. Apparently the repertoire is controlled at the level of gene expression. Preliminary data indicate that the Ly49A gene is hypomethylated in Ly49A+ NK cells but not other NK cells. Recently, we have demonstrated that Ly49 gene family members are subject to allelic exclusion, a phenomenon limited to extremely few mammalian genes. Additional preliminary data demonstrate that MHC class I genes influence the repertoire of Ly49 receptors expressed by NK cells. Finally, we have prepared mice which express an Ly49A cDNA transgene in all their NK cells; preliminary characterization of such mice suggests an influence of the transgene on the endogenous repertoire of Ly49 receptors. An understanding of the mechanisms that impose allelic exclusion of Ly49 family genes, and that account for MHC-dependent effects on the expression of Ly49 family members in different NK subsets, is fundamental to understanding how this lymphocyte repertoire is generated and regulated. The first specific aim is identify cis-acting DNA elements that regulate Ly49 family genes in different subsets and from different alleles. With the use of YAC and conventional genomic clones the complex will be mapped. DNase I hypersensitive sites will be mapped to identify putative regulatory elements, and the activities of these elements will be analyzed by transient tranfection experiments and transgenesis. The second aim is to unravel the mechanisms underlying allelic exclusion of Ly49 genes. The stability and completeness of allelic exclusion will be assessed, and the possible role of feedback regulation determined. The third specific aim is to determine whether class I molecules regulate the Ly49 repertoire, and whether a selection mechanism plays a role in the process.With these diverse approaches, elements associated with subset-specific and allele- specific expression may be identified, and models of how Ly49 genes are regulated by the MHC environment can be tested.